Factor Xa is a proteolytic enzyme which is formed in the blood coagulation cascade by activation of the proenzyme factor X and which, together with phospholipid and calcium ions, proteolytically splits factor II (prothrombin) at two points of the peptide chain and converts the said factor into factor IIa (thrombin) which finally causes coagulation. In certain pathological disorders, e.g. liver diseases, vitamin deficiency, etc., and in the dicoumarol therapy, the formation of factor X is reduced. In hereditary disturbances in the synthesis of factor X the formation of factor Xa, of course, also reduced correspondingly. Therefore, it is important to have at one's disposal a direct enzymatic assay method which allows factor Xa to be assayed photometrically in blood plasma in a simple and accurate manner.
The main methods for assaying factor Xa are the following:
(a) Biological assay method [cf. "Thrombosis and Bleeding Disorders," Nils U. Bang, Georg Thieme Verlag, p. 196 (1971)]: Factor X is activated to factor Xa by means of venom of Russel viper and calcium ions. In a one-step operation prothrombin is activated to thrombin by factor Xa in the presence of factor V and phospholipids, and thrombin converts indicator fibrinogen into fibrin. The clotting time is measured. The required factors II and V and fibrinogen are supplied by a substrate which is free from factor X. The clotting time is influenced by the degree of activation of factor X. The activation degree, under otherwise constant conditions, is a function of the concentration of factor X in the sample. This biological method allows no more than a rough assay to be carried out since the clotting time is read off subjectively by the experimentator. Furthermore, manipulated plasma is required during the preparation of which mistakes can occur. Moreover, the fibrinogen which is acting as an indicator is not formed directly, but via activated thrombin (indirect method). PA1 (b) Biochemical method [cf. "Thrombosis and Bleeding Disorders," N. U. Bang, Georg Thieme Verlag, p. 196/7 (171)]: If the factor X preparations to be tested are sufficiently pure, a more accurate assay method can be applied. Esnouf and Williams (1962) have shown that factor Xa has an esterase activity and hence splits synthetic amino acid esters. However, 50 to 100 .mu.g of factor X are required for this assay. On the other hand, lower concentrations of factor Xa can be determined by using carbobenzoxy-phenylalanine p-nitrophenyl ester and measuring the quantity of p-nitrophenol released per time unit. This assaying method has the following disadvantages: The ester used undergoes an autohydrolysis at the applied pH of 8 and, moreover, is not specific for factor Xa since it responds also to many other enzymes. The ester is not soluble in water so that acetone is required. As a result of all these disadvantages this assaying method is inaccurate and costly. PA1 (c) The published German patent application OS No. 25 52 570 discloses tetrapeptide derivatives which are intended to be used as substrates for assaying factor Xa. Bz-Ile-Glu-Gly-Arg-pNA.HCl is disclosed as an example of a tetrapeptide derivative which is split by factor Xa with formation of p-nitroaniline. The formation of p-nitroaniline can be followed spectrophotometrically. This method of assaying factor Xa is somewhat more accurate than the above-described biological and biochemical assaying methods. PA1 R.sup.2 represents a straight-chained or branched alkyl radical having 1 to 6, preferably 1 to 4, carbon atoms, a hydroxyalkyl radical having 1 to 2 carbon atoms, an alkoxyalkyl radical having 1 to 2 carbon atoms in the alkyl and 1 to 4 carbon atoms in the alkoxy, a benzyloxyalkyl radical having 1 to 2 carbon atoms in the alkyl, an .omega.-carboxyalkyl or .omega.-alkoxycarbonylalkyl radical which has 1 to 3 carbon atoms in the alkyl and the alkoxy group of which is straight-chained or branched and has 1 to 4 carbon atoms, an .omega.-benzyloxycarbonylalkyl radical having 1 to 3 carbon atoms in the alkyl, or a cyclohexyl-, cyclohexylmethyl-, 4-hydroxycyclohexylmethylphenyl-, benzyl-, 4-hydroxybenzyl- or imidazol-4-yl-methyl radical, PA1 R.sup.3 represents hydrogen or a straight-chained or branched alkyl radical having 1 to 4 carbon atoms, PA1 R.sup.4 represents hydrogen or a methyl or ethyl radical, and PA1 R.sup.5 represents an amino group which is substituted with aromatic or heterocyclic radicals and which is capable of being split off hydrolytically with formation of a coloured or fluorescent compound H-R.sup.5. PA1 (1) The chromogenic group R.sup.5 is attached to the carboxy group of the C-terminal arginine, whilst its .alpha.-amino group is protected by a protective group, e.g. a carbobenzoxy or tert.-butoxycarbonyl group, and the .delta.-guanidyl group of arginine is protected by protonation, e.g. with HCl, nitration or tosylation. The C-terminal group R.sup.5 -serves also as a protective group during the stepwise building up of the peptide chain. The remaining protective groups can be removed selectively as needed in order to attach the next amino acid derivatives until the desired peptide chain is completely built up. Finally, the remaining protective groups can be entirely removed without group R.sup.5 -being affected (cf. e.g. Miklos Bodansky et al., "Peptide Synthesis," Interscience Publishers, p. 163-165, 1966). PA1 (2) First of all, the peptide chain is built up (according to Bodansky, loc. cit.) whilst the C-terminal carboxyl group of arginine is protected by a usual ester group, e.g. a methoxy, ethoxy or benzyloxy group. The ester groups can be removed by alkaline hydrolysis, except for the tert.-butoxy group which has to be removed selectively by means of trifluoroacetic acid. If the .delta.-guanidyl group of arginine is protonated, the said ester group is removed by trypsin, no racemization taking place in this case. Thereafter, the chromogenic group R.sup.5 -is introduced. If the .delta.-guanidino group of arginine is protected by a nitro or tosyl group and the N-terminal .alpha.-amino group of the tripeptide derivative is protected by a carbobenzoxy group or a p-methyl, p-methoxy or p-chloro-benzyloxycarbonyl group, or a tert.-butoxy group, all these protective groups are removed simultaneously. The removal can be achieved by treating the protected tripeptide derivative with anhydrous HF at room temperature, and as a result all the above-mentioned amino and .delta.-guanidino protective groups are removed. The removal can also be carried out by treatment with 2 N HBr in glacial acetic acid at room temperature if the protected tripeptide derivative does not contain any nitro or tosyl as protective groups. PA1 D-Aadi=D-.alpha.-amino-adipic acid PA1 Ac=acetyl PA1 Ac.sub.2 O=acetic anhydride PA1 AcOH=acetic acid PA1 Ala=L-alanine PA1 D-Ala=D-alanine PA1 AOA=D-.alpha.-amino-octanoic acid PA1 Arg=L-arginine PA1 D-Asp=D-aspartic acid PA1 BOC=tert.-butoxycarbonyl PA1 Bu=butyl PA1 But=L-2-aminobutyric acid PA1 D-But=D-2-aminobutyric acid PA1 Bz=benzoyl PA1 Bzl=benzyl PA1 Bz.sub.2 O=benzoic anhydride PA1 ChA=quinonyl amide PA1 D-CHA=D-3-cylohexylalanine PA1 D-CHG=D-2-cyclohexylglycine PA1 D-CHT=D-3-(4-hydroxycyclohexyl)-alanine=tyrosine substituted in the nucleus PA1 Cbo=carbobenzoxy PA1 DMF=dimethylformamide PA1 DPA=dimethyl ester of 5-amido-isophthalic acid PA1 TLC=thin layer chromatography or thin layer chromatogram PA1 Et=ethyl PA1 Et.sub.3 N=triethylamine PA1 Gly=glycine PA1 D-Glu=D-glutamic acid PA1 D-His=D-histidine PA1 HMPTA=N,N,N',N',N",N"-hexylmethyl-phosphoric acid triamide PA1 D-Ile=D-isoleucine PA1 D-Leu=D-leucine PA1 SS=solvent system PA1 MCA=7-amido-4-methylcoumarin PA1 MeO=methoxy PA1 MeOH=methanol PA1 NA=naphthylamide PA1 D-Nleu=D-norleucine PA1 D-Nval=D-norvaline PA1 OtBu=tert.-butoxy PA1 OpNP=p-nitrophenoxy PA1 pNA=p-nitroanilide PA1 Pr=propyl PA1 D-Ph'Gly=D-2-phenylglycine PA1 D-Phe=D-phenylalanine PA1 Sar=sarkosine=N-methylglycine PA1 D-Ser=D-serine PA1 TFA=trifluoroacetic acid PA1 THF=tetrahydrofuran PA1 D-Thr=D-threonine PA1 Tos=p-toluenesulfonyl PA1 D-Tyr=D-tyrosine PA1 D-Val=D-valine
However, the tetrapeptide derivatives described in German patent application DE-OS No. 25 52 570 are not sufficiently soluble in aqueous media to allow the assay of factor Xa to be carried out at substrate saturation. In the case where extremely low concentrations of factor Xa have to be determined, e.g. in pathological plasma, the said tetrapeptide derivatives are not sufficiently sensitive to allow reasonably accurate measuring values to be obtained. If the quantity of factor Xa to be measured were increased by adding a further quantity of plasma, a precipitation of the tetrapeptide substrate would take place under the influence of plasmaproteins, and as a result it would be impossible to perform the enzyme assay.